High‐risk neuroblastoma (NB) is responsible for a disproportionate number of childhood deaths due to cancer. One indicator of high‐risk NB is amplification of the neural MYC ( MYCN ) oncogene, which is currently therapeutically intractable. Identification of anaplastic lymphoma kinase (ALK) as an NB oncogene raised the possibility of using ALK tyrosine kinase inhibitors (TKIs) in treatment of patients with activating ALK mutations. 8–10% of primary NB patients are ALK‐positive, a figure that increases in the relapsed population. ALK is activated by the ALKAL2 ligand located on chromosome 2p, along with ALK and MYCN , in the “2p‐gain” region associated with NB. Dysregulation of ALK ligand in NB has not been addressed, although one of the first oncogenes described was v‐sis that shares > 90% homology with PDGF. Therefore, we tested whether ALKAL2 ligand could potentiate NB progression in the absence of ALK mutation. We show that ALKAL2 overexpression in mice drives ALK TKI‐sensitive NB in the absence of ALK mutation, suggesting that additional NB patients, such as those exhibiting 2p‐gain, may benefit from ALK TKI‐based therapeutic intervention.

High-risk neuroblastoma (NB) often involvesMYCNamplification as well as mutations inALK. Currently, high-risk NB presents significant clinical challenges, and additional therapeutic options are needed. Oncogenes likeMYCNandALKresult in increased replication stress in cancer cells, offering therapeutically exploitable options. We have pursued phosphoproteomic analyses highlighting ATR activity in ALK-driven NB cells, identifying the BAY1895344 ATR inhibitor as a potent inhibitor of NB cell growth and proliferation. Using RNA-Seq, proteomics and phosphoproteomics we characterize NB cell and tumour responses to ATR inhibition, identifying key components of the DNA damage response as ATR targets in NB cells. ATR inhibition also produces robust responses in mouse models. Remarkably, a 2-week combined ATR/ALK inhibition protocol leads to complete tumor regression in two independent genetically modified mouse NB models. These results suggest that NB patients, particularly in high-risk groups with oncogene-induced replication stress, may benefit from ATR inhibition as therapeutic intervention.

Activation of the RAS-RAF-MEK-ERK signaling pathway is implicated in driving the initiation and progression of multiple cancers. Several inhibitors targeting the RAS-MAPK pathway are clinically approved as single- or polyagent therapies for patients with specific types of cancer. One example is the MEK inhibitor trametinib, which is included as a rational polytherapy strategy for treating EML4-ALK–positive, EGFR-activated, or KRAS-mutant lung cancers and neuroblastomas that also contain activating mutations in the RAS-MAPK pathway. In addition, in neuroblastoma, a heterogeneous disease, relapse cases display an increased rate of mutations in ALK, NRAS, and NF1, leading to increased activation of RAS-MAPK signaling. Co-targeting ALK and the RAS-MAPK pathway is an attractive option, because monotherapies have not yet produced effective results in ALK-addicted neuroblastoma patients. We evaluated the response of neuroblastoma cell lines to MEK-ERK pathway inhibition by trametinib. In contrast to RAS-MAPK pathway-mutated neuroblastoma cell lines, ALK-addicted neuroblastoma cells treated with trametinib showed increased activation (inferred by phosphorylation) of the kinases AKT and ERK5. This feedback response was mediated by the mammalian target of rapamycin complex 2–associated protein SIN1, resulting in increased survival and proliferation that depended on AKT signaling. In xenografts in mice, trametinib inhibited the growth of EML4-ALK–positive non–small cell lung cancer and RAS-mutant neuroblastoma but not ALK-addicted neuroblastoma. Thus, our results advise against the seemingly rational option of using MEK inhibitors to treat ALK-addicted neuroblastoma.

High-risk neuroblastoma (NB) is a significant clinical challenge. MYCN and Anaplastic Lymphoma Kinase (ALK), which are often involved in high-risk NB, lead to increased replication stress in cancer cells, suggesting therapeutic strategies. We previously identified an ATR (ataxia telangiectasia and Rad3-related)/ALK inhibitor (ATRi/ALKi) combination as such a strategy in two independent genetically modified mouse NB models. Here, we identify an underlying molecular mechanism, in which ALK signaling leads to phosphorylation of ATR and CHK1, supporting an effective DNA damage response. The importance of ALK inhibition is supported by mouse data, in which ATRi monotreatment resulted in a robust initial response, but subsequent relapse, in contrast to a 14-d ALKi/ATRi combination treatment that resulted in a robust and sustained response. Finally, we show that the remarkable response to the 14-d combined ATR/ALK inhibition protocol reflects a robust differentiation response, reprogramming tumor cells to a neuronal/Schwann cell lineage identity. Our results identify an ability of ATR inhibition to promote NB differentiation and underscore the importance of further exploring combined ALK/ATR inhibition in NB, particularly in high-risk patient groups with oncogene-induced replication stress.

Neuroblastoma is the most commonly diagnosed extracranial tumor in the first year of life. Approximately 9% of neuroblastoma patients present germline or somatic aberrations in the gene encoding for anaplastic lymphoma kinase (ALK). This increases in high-risk neuroblastomas, which have a 14% frequency of ALK aberrations at the time of diagnosis and show increasing numbers at relapse. Abrogating ALK activity with kinase inhibitors is employed as clinical therapy in malignancies such as non-small cell lung cancer and has shown good results in pediatric inflammatory myofibroblastic tumors and anaplastic large cell lymphomas. A phase I clinical trial of the first generation ALK inhibitor, crizotinib, in neuroblastoma patients showed modest results and suggested that further investigation was needed. Continuous development of ALK inhibitors has resulted in the third generation inhibitor repotrectinib (TPX-0005), which targets the active kinase conformations of ALK, ROS1 and TRK receptors. In the present study we investigated the effects of repotrectinib in a neuroblastoma setting in vitro and in vivo. Neuroblastoma cell lines were treated with repotrectinib to investigate inhibition of ALK and to determine its effect on proliferation. PC12 cells transfected with different ALK mutant variants were used to study the efficacy of repotrectinib to block ALK activation/signaling. The in vivo effect of repotrectinib was also analyzed in a neuroblastoma xenograft model. Our results show that repotrectinib is capable of inhibiting signaling activity of a range of ALK mutant variants found in neuroblastoma patients and importantly it exhibits strong antitumor effects in a xenograft model of neuroblastoma.

High‐risk neuroblastomas exhibit a high degree of intratumoral heterogeneity. Single‐cell RNA sequencing has greatly improved our understanding of these tumors, but the method lacks cellular tissue context and spatial information about local signaling dynamics. To address this, we profiled untreated and chemotherapy‐treated high‐risk neuroblastomas from archived, formalin‐fixed, paraffin‐embedded (FFPE) tissues from two patients using spatial transcriptomics. We confirmed the transcriptional and cellular heterogeneous nature of the neuroblastoma microenvironment and identified several unique spatial niches and patterns. In one of the treated tumors, a spatially constrained cluster of undifferentiated and 11p‐gained cancer cells was identified, surrounded by a rim of macrophages. A signaling interaction between the chemokine CCL18 and its receptor PITPNM3 was predicted between these cells. In the other tumor, we identified a stromal cluster with high transcriptional similarity to the adrenal cortex. These adrenocortical‐like cells expressed several oncogenic ligand‐encoding genes (e.g. ALKAL2 and NRTN), which were predicted to communicate with neighboring cancer cells that expressed the corresponding receptors (e.g. ALK, RET). Several of these interactions were further validated experimentally and were shown to be clinically relevant. Collectively, our spatial analysis identifies multiple previously unrecognized signaling axes that may offer novel therapeutic options in neuroblastoma. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.